Sunday, March 15, 2015

Stem Cells
(SC) can differentiate into many different cell types, offering the potential
of replacing failing cells, tissues, or even entire organs with new ones
generated from the patient’s own or related donor SC (National Academy of
Sciences Workshop summary).To be useful for
therapy in the clinic, it would be necessary to grow and expand SCs in culture.

The authors explored the proliferation of human embryonic SC
(HESC), which are prepared from disrupted embryos, and the less-controversial human
inducible pluripotent stem cells (hiPSCs), which can be prepared from several
adult tissues, including blood, skin, and fat. They obtained 1 HESC line, WA09, from the
WiCell Research Institute and they generated 3 hiPSC lines from fetal dermal
fibroblasts by over-expressing the ‘standard reprogramming factors’ (pluripotency-conferring
genes, transduced OCT4/POU5F1, SOX2, KLF4, and MYC).

They compared four standard SC culture conditions: with or
without a feeder cell layer and enzymatic or “mechanical” (dissection)
disruption, with 6 replicate cultures per condition, for over 100 “passages”
(transfers to fresh cultures). Previous studies cited here revealed genomic
changes (small duplications) that are not detectable by karyotyping,
particularly on chromosome 12, where the pluripotency-related gene NANOG is
encoded, and chromosome 20, where the survival gene Bcl-xL is encoded.In addition to measuring proliferation, telomere
length, pluripotency by teratoma formation, they also analyzed over a million
reference SNPs around the genome and used those SNPs to assess copy number
variation (CNV).

Not surprisingly, genomic changes increased with
time in culture, both in aberration number (A) and total length (B) (Figure 2, shown,
WA09 HESC: left duplications and right deletions).The number of aberrations was lowest in
“EcmMech” condition, i.e. cultures without feeder cells (only extracellular
matrix, ECM), and disrupted mechanically (blue line). The number and length of aberrations was worst
with MefEnz (green line), cultured with feeder cells (mouse embryo fibroblasts,
Mef) and disrupted enzymatically.They
conclude that there is a “need for careful assessment of the effects of culture
conditions on cells intended for clinical therapies”.

Mission

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